MINFLUX fluorescence nanoscopy in biological tissue

Thea Moosmayer, Kamila A Kiszka, Volker Westphal, Jasmin K Pape, Marcel Leutenegger, Heinz Steffens, Seth G N Grant, Steffen J Sahl, Stefan W Hell

Research output: Contribution to journalArticlepeer-review

Abstract

Optical imaging access to nanometer-level protein distributions in intact tissue is a highly sought-after goal, as it would provide visualization in physiologically relevant contexts. Under the unfavorable signal-to-background conditions of increased absorption and scattering of the excitation and fluorescence light in the complex tissue sample, superresolution fluorescence microscopy methods are severely challenged in attaining precise localization of molecules. We reasoned that the typical use of a confocal detection pinhole in MINFLUX nanoscopy, suppressing background and providing optical sectioning, should facilitate the detection and resolution of single fluorophores even amid scattering and optically challenging tissue environments. Here, we investigated the performance of MINFLUX imaging for different synaptic targets and fluorescent labels in tissue sections of the mouse brain. Single fluorophores were localized with a precision of <5 nm at up to 80 µm sample depth. MINFLUX imaging in two color channels allowed to probe PSD95 localization relative to the spine head morphology, while also visualizing presynaptic vesicular glutamate transporter (VGlut) 1 clustering and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) clustering at the postsynapse. Our two-dimensional (2D) and three-dimensional (3D) two-color MINFLUX results in tissue, with <10 nm 3D fluorophore localization, open up broad avenues to investigate protein distributions on the single-synapse level in fixed and living brain slices.

Original languageEnglish
Pages (from-to)e2422020121
JournalProceedings of the National Academy of Sciences of the United States of America
Volume121
Issue number52
DOIs
Publication statusPublished - 20 Dec 2024

Keywords / Materials (for Non-textual outputs)

  • Animals
  • Mice
  • Microscopy, Fluorescence/methods
  • Brain/diagnostic imaging
  • Fluorescent Dyes/chemistry
  • Nanotechnology/methods
  • Optical Imaging/methods
  • Receptors, AMPA/metabolism

Fingerprint

Dive into the research topics of 'MINFLUX fluorescence nanoscopy in biological tissue'. Together they form a unique fingerprint.

Cite this